The overall goal of this proposal is to understand how mammalian cells detect and respond to the presence of intracellular bacterial pathogens. Despite antibiotics, bacterial infections continue to present a significant public health challenge. Our studies utilize the gram-negative bacterium Legionella pneumophila, the causative agent of a severe pneumonia called Legionnaires' Disease, as a model for understanding how bacterial pathogens interact with macrophages. The virulence of Legionella depends on its ability to survive and grow within macrophages. Previous work has established that two genes (Naip5 and Ipaf) are instrumental in orchestrating cellular defenses that protect macrophages from Legionella infection, but the molecular mechanism by which Naip5/Ipaf confer resistance to Legionella has remained largely mysterious. Naip5 and Ipaf exhibit homology to a large family of cytosolic pathogen- detector proteins called the Nod-like proteins. Our preliminary results suggest that resistance to Legionella depends on rapid triggering of a Naip5/Ipaf-containing inflammasome that occurs upon the detection of bacterial flagellin in the macrophage cytosol. Inflammasome activation is connected to a variety of human diseases, and thus use of Legionella as a model for understanding inflammasome activation will have broad implications for our understanding of human health and disease. We have also made the unexpected observation that Naip/Ipaf are not sufficient to protect macrophages from Legionella, and that in addition, signaling via the tumor necrosis factor receptor is also required. TNF is already an important therapeutic target in the clinical treatment of diseases such as arthritis and Crohn's Disease. Thus, a deeper understanding of the molecular basis by which Naip/Ipaf and TNF collaborate to restrict Legionella growth could possibly be of great relevance to human health and disease. Thus, the specific aims of this grant proposal are: 1. Test the hypothesis that the intracellular presence of bacterial flagellin protein is sufficient to trigger the Ipaf/Naip5-dependent signaling pathways that restrict bacterial growth; map the determinants within flagellin required to trigger Ipaf/Naip5; and using this information, test the hypothesis that flagellin physically interacts with Naip5 and/or Ipaf. 2. Test the hypothesis that Naip5 is critical for macrophage resistance to Legionella by targeted deletion of Naip5 in mice. 3. Test the hypothesis that Naip5/Ipaf signaling protects macrophages by synergizing with TNF signaling. PUBLIC HEALTH RELEVANCE: It is anticipated that results obtained from the above work will permit a deeper understanding of how bacteria cause disease and of what factors lead to successful immune responses to these bacteria. Such knowledge should contribute to rational approaches to designing novel antibacterial therapies.